The invention relates to modified oligonucleotides useful in technologies which rely on complementarity or specificity of oligomer sequences for drug delivery or for direct interference with nucleic acid activity. More specifically, the invention concerns oligomers derivatized at the 2xe2x80x2 position, which are stable to nuclease activity.
There has been considerable activity in recent years concerning the design of nucleic acids as diagnostic and therapeutic tools. One aspect of this design relies on the specific attraction of certain oligomer sequences for nucleic acid materials in vivo which mediate disease or tumors. This general approach has often been referred to as xe2x80x9canti-sensexe2x80x9d technology. An oversimplified statement of the general premise is that the administered oligomer is complementary to the DNA or RNA which is associated with, and critical to, the propagation of an infectious organism or a cellular condition such as malignancy. The premise is that the complementarity will permit binding of the oligomer to the target nucleic acid, thus inactivating it from whatever its ordinary function might have been.
A simple illustration would be the administration of a DNA oligomer complementary to an mRNA which encodes a protein necessary to the progress of infection. This administered DNA would inactivate the translation of the mRNA and thus prevent the formation of the protein. Presumably the DNA could be directly administered, or could be used to generate an mRNA complement to the target mRNA in situ. There is by now extensive literature concerned with this general approach, and the methods of utilizing oligomers of this type which are complementary to target RNA or DNA sequences are set forth, for example, in van der Krol, A. R., et al., Biotechniques (1988) 6:958-976; Stein, C. A., et al., Cancer Research (1988) 48:2659-2668; Izant, J. G., et al., Science (1985) 229:345-352; and Zon, G., Pharmaceutical Research (1988) 5:539-549, all incorporated herein by reference. In addition, a bibliography of citations relating to anti-sense oligonucleotides has been prepared by Dr. Leo Lee at the Frederick Cancer Research Facility in Frederick, Md.
There are two conceptual additions to the general idea of using complementarity to interfere with nucleic acid functionality in vitro. The first of these is that strict complementarity in the classical base-pairing sense can be supplemented by the specific ability of certain oligonucleotide sequences to recognize and bind sequences in double-helical DNA and to insert itself into the major groove of this complex. A fairly recent but reasonably definitive series of papers has elucidated the current rules for such specificity. These papers take account of very early work by, for example, Arnott, S., et al., J Mol Biol (1974) 88:509-521, which indicates the general principle of binding as triplexes poly-dT/poly-dA/poly-dT, and the corresponding analogous triplex involving poly-dC as summarized by Moser, H. E., et al., Science (1987) 238:645-650. More recent studies show that the earlier rule (which was that recognition could be achieved by a homopyrimidine oligomer to homopurine/homopyrimidine stretches in the duplex) could be extended to patterns whereby mixed sequences can also be recognized (Griffin, L. C., et al., Science (1989) 245:907-971. Further summaries of these phenomena are given, for example, in a review article by Maher III, L. J., et al., Science (1989) 245:725-730. Additional related disclosures of triple-helix formation are those by Cooney, M., et al., Science (1988) 241:456-459; Francois, J.-C., Nucleic Acids Res (1988) 16:11431-11440; and Strobel, S. A., et al., J Am Chem Soc (1988) 110:7927-7929. While further details are needed to provide exact sequence specificity studies in this context, it is clear that the rules for xe2x80x9ccomplementarityxe2x80x9d in this sense of specific embedding into the major groove of the double-helix are rapidly emerging.
The second aspect of anti-sense technology which deviates from the simple concept of base-pair complementarity in native oligonucleotides results from the early recognition that oligonucleotides, especially RNAs, are highly susceptible to nuclease cleavage in biological systems. In order for these materials to remain active drugs, it would be necessary to stabilize the administered oligonucleotides against this degradation. The approach that has so far been used has been to modify the phosphodiester linkages so as to be resistant to attack by these enzymes. In particular, the phosphodiester linkage has been replaced by phosphoramidate linkages, methylphosphonate linkages, and phosphorothioate linkages. These approaches have certain results with regard to stereoisomerism and its associated impact on hybridization to the target sequences that make them less than completely satisfactory. An alternate approach has been to modify the nucleosides by using 2xe2x80x2-O-methyl ribose or the alpha-anomers of the conventional nucleoside residues. In addition, oligomers containing 2xe2x80x2 amino groups have been prepared via their triphosphate analogs and enzyme-catalyzed polymerization by Hobbs, J., et al., Biochemistry (1973) 12:5138-5145. Some of these approaches have been summarized in the Zon review cited in the previous paragraph.
The present invention provides additional 2xe2x80x2-substituted pentose moieties for inclusion in the oligomers useful in this technology which are resistant to nuclease activity, and may optionally be combined with additional modifications such as those set forth above.
The invention is directed to nucleosides and nucleotides of the formula: 
wherein
B is a purine or pyrimidine residue or analog thereof;
W1 is H, (PO3)mxe2x88x922 wherein m is an integer of 1-3; a protecting group, or a group reactive to link hydroxyl groups;
W2 is H, PO3xe2x88x922, a protecting group, or a group reactive to link hydroxyl groups;
X is O, S, NR or CR2 wherein each R is independently H or alkyl (1-6 C);
Y is a linker moiety, a drug residue optionally attached through a linker moiety, a label optionally attached through a linker moiety, or a property-affecting residue optionally attached through a linker moiety, wherein said Xxe2x80x94Y substituent renders an oligomer in which said nucleoside or nucleotide of formula (1) is included more stable to treatment with nuclease than said oligomer which incorporates a corresponding nucleotide having xe2x80x94H2 or xe2x80x94HOH at the 2-position.
These materials are useful as intermediates in the synthesis of the oligomers of the invention, which are oligomers of the formula: 
wherein each B is independently a purine or pyrimidine residue or analog thereof;
W3 and W4 are each independently H, PO3xe2x88x922, a protecting group, or a group reactive to link hydroxyl groups;
n is an integer of 1-200;
each Z is independently a nucleotide linking residue covalently conjugating the hydroxyl groups of sequential nucleotide residues;
each A is independently selected from the group consisting of H, OH, OH derivatized to a protecting group, and Xxe2x80x94Y wherein
X is O, S, NR, or CR2 wherein each R is independently H or alkyl (1-6 C); and
Y is a linker moiety, a drug residue optionally attached through a linker moiety, a label optionally attached through a linker moiety, or a property-affecting group optionally attached through a linker moiety;
wherein at least one A is Xxe2x80x94Y; and
wherein the oligomer is more stable to nuclease than the Corresponding oligomer wherein all A are H or OH.
The oligomeric materials of formula (2) are useful as therapeutic or prophylactic agents in protocols which are directed against infectious disease or malignancy by targeting specific DNA and/or RNA sequences associated with the condition, as well as in diagnostic applications.
A. Definitions
The oligomers of the invention contain the residue of at least one nucleotide of formula (1). In this formula, and in the oligomers, B represents a conventional purine or pyrimidine base such as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U) or protected forms thereof. Suitable protecting groups include acyl, isobutyryl, benzoyl, and the like. B can, however, also represent a modified or protected form or related derivative of these conventionally occurring bases, i.e., an xe2x80x9canalog.xe2x80x9d A wide range of these analogous heterocyclic bases is known in the art. For example, commonly encountered among such analogs are those which include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thioridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5xe2x80x2-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine.
As the modification of these bases affects both the stability of the resulting oligomer and the hybridization ability of the sequence, in many instances only a limited number of such substitutions in a particular oligomer is desirable. However, there are other instances when an entire oligomer may be composed of nucleotide residues containing an analog. For example, DNA polymers of uridine and oligomers of 5-bromouridine, and 5-methyl cytidine have been shown to be therapeutically and diagnostically useful. As will be apparent to practitioners of the art, a sensible approach must be used in designing oligomers containing either conventional or modified base forms so that the properties of the resulting monomer are in the desired range. Therefore, in some cases, less than 10% of the bases indicated as xe2x80x9cBxe2x80x9d in the sequence of formula (2) will be replaced by analogous bases, preferably less than 5%, more preferably less than 1%, and most preferably none at all. However, in other cases, complete replacement by analogs is desirable.
Similar comments apply to the substitution for the bases of nonfunctional substituents such as alkyl or aryl nonheterocyclic groups; however, such substitutions may be permissible to a highly limited extent, for example one residue per 20 or so without actually destroying the functionality of the oligomer. It does not appear there is any particular advantage in making these replacements, and they are permissible only because the replacement may be overwhelmed by the functionality of the remainder of the molecule.
Substituents designated W1-W4 may be H, PO3xe2x88x922, (PO3)mxe2x88x922, protecting groups, or groups reactive to link hydroxyl group.
A xe2x80x9cprotecting groupxe2x80x9d in the context of W is a substituent which prevents the reactivity of the xe2x80x94OH to which it is bound in a chemical reaction, typically a reaction to link sequential nucleotides, and which can be removed when the reaction is completed. Typical protecting groups in the compounds of the invention include 4,4xe2x80x2-dimethoxy trityl (DMT), 4-monomethoxytrityl and trityl.
A xe2x80x9cgroup reactive to link hydroxylsxe2x80x9d is an intermediate residue in the formation of an internucleotide link between the 5xe2x80x2 and 3xe2x80x2 hydroxyls. Thus, the reaction of said group with the appropriate xe2x80x94OH of the adjacent nucleotide results in the nucleotide linking residue, Z.
The linking residue represented by Z is typically P(O)Oxe2x88x92 in naturally occurring oligonucleotides, but can also be P(O)S, P(O)NR2, P(O)R, or P(O)ORxe2x80x2, or can be CO or CNR2 wherein R is H or alkyl (1-6C) and Rxe2x80x2 is alkyl (1-6C) or can be xe2x80x94CX2xe2x80x94 wherein each X is independently an electron-withdrawing substituent as described in copending application attorney docket 4610-0005, filed on even date herewith, assigned to the same assignee and incorporated herein by reference. In general, Z can be any nucleotide linking moiety conventionally used to conjugate nucleotide residues to form oligonucleotides.
A linker moiety, as represented by Y (Yxe2x80x2 when covalently bound to an additional substituent), is any bivalent bridging residue used to attach a desired substituent to the monomer or oligomer. The linker may be simply binding methylene groupsxe2x80x94i.e., xe2x80x94(CH2)nxe2x80x94 or may include heteroatoms and functional groups, e.g., xe2x80x94CH2OCH2CH2Oxe2x80x94 or xe2x80x94CH2Oxe2x80x94CH2CH2NHxe2x80x94 or xe2x80x94COOCH2CH2Oxe2x80x94. The linker residue may also be a residue derived from a commercially available bifunctional linker such as the hetero- and homo-bifunctional linkers marketed by Pierce Chemical Co., Rockford, Ill.
A xe2x80x9cdrug residue,xe2x80x9d represented by Y, is the attached portion of a drug useful in conjunction with the oligomer, such as a drug capable of intercalation or of insertion into the minor groove of a DNA-DNA or DNA-RNA double helix or which can effect oligonucleotide cleavage. Examples of such drugs are set forth hereinbelow.
A xe2x80x9clabel residuexe2x80x9d is the attached portion of a label such as a moiety containing a radioisotope, a fluorophore, a chromophore, an enzyme and the like. Such labels may be desirable if this oligomer is to be used in diagnosis.
A xe2x80x9cproperty-affectingxe2x80x9d residue is a residue which, by virtue of its presence, results in changed properties of the oligomer. Such changed properties include, but are not limited to, enhancement of cell permeation properties, enhancement of the ability of the oligomer to hybridize to or otherwise bind to oligonucleotide sequences and enhancement of stability to nucleases.
Compounds of the invention that contain groups which are negatively charged at neutral pH can be prepared as their salts. The salts are formed from inorganic bases, such as NaOH, KOH or Ca(OH)2, or organic bases, such as caffeine, various alkylamines, TEA, and DBU.
Compounds of the invention that contain groups which are positively charged at neutral pH can be prepared as acid-addition salts formed from inorganic acids such as HCl, H2SO4 or H3SO4, or from organic acids such as acetic, succinic or citric.
Thus, the nucleotides and their corresponding oligonucleotides may exist as salts depending on the pH at which they find themselves or at which they are prepared. The phosphate and phosphodiester moieties associated with these molecules permits the formation of basic salts such as those formed from inorganic ions such as sodium, potassium, ammonium ions, but especially divalent ions such as calcium and magnesium ions. It is possible, but less common, to form salts of these materials with organic bases such as organic amines or heterocycles.
The invention compounds differ from those of the prior art by having in the 2xe2x80x2 position, at either chirality, a substituent which confers nuclease stability and, optionally, provides the capacity to deliver a drug, for example, a reagent which is effective to interact with duplex DNA in its minor groove, provides a label, or provides some additional property. While the remainder of the molecule in formula (2) is sometimes shown for convenience as having the features of the native oligonucleotides, and, indeed, this is often the most preferred embodiment, also included within the invention are molecules which contain the 2xe2x80x2 extensions and substitutions of the invention, but also contain additional modifications such as replacement of one or more of the phosphodiester linkages with, for example, a phosphorothioate or methyl phosphonate linkage; a phosphoramidate linkage, including those containing organic amino substituents, such as morpholidates, replacement of all beta-anomers by the alpha-anomer, and the presence or absence of protecting groups or phosphate residues at the 5xe2x80x2- and 3xe2x80x2-termini.
At the 2xe2x80x2 position, the invention discloses several general categories of substituents, which share a common type of linkage to the 2xe2x80x2 carbon through a substituent selected from O, S, R and CR2, wherein each R is independently selected. In all embodiments, Xxe2x80x94Y represents a substituent which is capable, by virtue of its presence, of inhibiting the cleavage of the oligomer in which it is included by nucleases. All of the oligomers of the invention are relatively stable to nucleases.
The stability of the oligomers to nucleases can be determined using any convenient assay, but is conveniently assessed using the snake venom assay illustrated hereinbelow. This assay is conducted as follows: The assay buffer is 0.5 M Tris HCl, pH 8.0, containing 100 xcexcM/MgCl2. Commercially available phosphodiesterase isolated from Croatalus durissus is obtained from Boehringer Mannheim as a 50% (v/v) solution in glycerol, pH 6, with a specific activity of approximately 1.2 U/mg. One ul of the phosphodiesterase-containing solution is added to 100 ul buffer, and oligomers are tested by reconstituting 0.15 OD of oligomer in the 100 ul buffer/venom prepared above. Degradation is monitored by observing the disappearance of the 260 nm absorption of the oligomer at its characteristic retention time on HPLC, and measuring the appearance of degradation products.
The oligomers of the invention which contain at least one nucleotide residue containing the 2xe2x80x2 substituent are more stable to nuclease as judged by the foregoing assay than the corresponding oligomer containing an unsubstituted 2xe2x80x2 position in place of the substituted positions in the invention compounds. By comparing the rate of hydrolysis in the snake venom assay with the invention compound, with that of the corresponding oligomer which is not derivatized in the 2xe2x80x2 position, it can be assessed whether the presence of the 2xe2x80x2 substituent(s) stabilized the oligomer to cleavage by nucleases.
Typical embodiments of Y, when its sole function is to alter the properties of the oligomer, include alkyl or alkenyl (2-20C), preferably 2-6C, which may or may not be substituted with noninterfering substituents, such as xe2x80x94OH, xe2x95x90O, carboxyl, halo, amino groups and the like, aryl or substituted aryl (6-20C), various alkyl silyl derivatives of the formula SiR3 (wherein each R is alkyl of 2-6C), and similar substituents which also contain heteroatoms. If Y includes a linker moiety, this portion of Y (Yxe2x80x2) will provide functional group(s) for conjugation to additional substances. For example, an embodiment of Yxe2x80x2 of 1-20C may contain a hydroxyl, amino, mercaptyl, carboxy, keto, or other functional group or several of these in combination. Typical examples include xe2x80x94CH2COOH; xe2x80x94CH2CONH2; xe2x80x94CH2COOEt; xe2x80x94CH2CONHCH2CH2NH2; and the like.
The linker moiety may be utilized to couple the nucleoside, nucleotide or residue within the oligomer to a reagent or drug, such as a drug which is known to interact with the minor groove of duplex DNA or DNA/RNA. A wide variety of these reagents and substances is known and the function, in vivo, is generally to inactivate the DNA duplex with which these reagents interact. Typical examples of such agents include netropsin and its derivatives, anthramycin, quinoxaline antibiotics, actinomycin, pyrrolo (1-4) benzodiazepine derivatives and intercalating agents.
Other drugs, besides those which seek the minor groove, may also be used. Intercalators, toxins, degradation inducers, and the like can also be used. Furthermore, the drug need not be linked through the linker moiety, but may be directly associated with the substituent X, depending on the chemistry of the particular drug.
Another embodiment of Y represents label optionally linked to X through a linker moiety, but also possibly directly attached, again depending on the chemistry in the particular case. Suitable labels include radioisotopes, fluorescent labels, such as fluoroscein and dansyl, chromophores, enzymes and the like. A wide variety of labels is known and can be used to provide detectability when the oligomers of the invention are used as probes or in other specific binding diagnostic assays.
Finally, Y can be a substituent which confers altered properties on the oligomer. It has already been noted that all of the substituents, including drugs and label, confer increased nuclease stability. However, additional properties may also be affectedxe2x80x94for example, agents which cleave associated nucleotide chains may be attached; cell permeation enhancement may occur by virtue of the substituent, or Y may enhance the hybridization of the oligomer to complementary oligonucleotides or to a DNA/DNA or DNA/RNA helix. As with all of the foregoing embodiments of Y, the activity or property-changing substituent may be directly bound to X or may be conjugated through a linker moiety.
B. Preparation of the Invention Compounds
Some of the compounds of the invention which are nucleosides or nucleotides are prepared by reacting the corresponding nucleotide or nucleoside having OH in the 2xe2x80x2 position with suitable reagents to effect conversion to the substituted form. In some cases, the 2xe2x80x2 substituent may be derived from cyclic forms of the nucleoside or nucleotide. Further conversions may be required, as illustrated below, to activate the nucleotide or nucleoside for inclusion into the oligomer. The techniques for these conversions are generally understood in the art, as are the techniques for sequential synthesis of the oligomers.
In particular, dimers may be synthesized to evaluate the effect of the 2xe2x80x2 substituent on nuclease activity. In the formation of the dimer, the converted nucleoside or nucleotide of the invention, protected at the 5xe2x80x2 position and containing a group reactive to link hydroxyl groups at the 3xe2x80x2 position, is reacted with, for example, thymidine or cytidine linked at the 3xe2x80x2 position to solid support and the resulting dimer is cleaved from the support and deprotected.
In all of the foregoing cases, conversions to change the functionality and character of the 2xe2x80x2 substituent can be conducted either at the monomer or oligomer level. Thus, a 2xe2x80x2 substituent which has the formula for Xxe2x80x94Y OCH2COOEt can be converted to an embodiment wherein the substituent is the free acid or the amide either when the ester is a substituent of the single nucleoside or nucleotide, of a dimer, or contained in the oligomeric chain.
The compounds of the invention which are oligomers are obtained by inclusion of the derivatized nucleotide or nucleoside into the oligomer using standard solid phase oligonucleotide synthesis techniques. Such techniques are commercially available for formation of both standard phosphodiester linkages and the conventional substitute linkages described above.
C. Utility and Administration
The compounds of the invention are useful in a manner known in the art for nuclease-inhibited, specifically complementary or binding, oligomers. As set forth above, the general methods for utilization of these compounds are known, and their application to specific diseases or conditions depends on the ascertainment of the appropriate binding specificity. The determination of this binding specificity does not affect the manner of preparation or application of the modified compounds of the invention.
Accordingly, the modified oligomers of the invention are useful in therapeutic, diagnostic and research contexts. In therapeutic applications, the oligomers are utilized in a manner appropriate for antisense therapy in generalxe2x80x94as described above, antisense therapy as used herein includes targeting a specific DNA or RNA sequence through complementarity or through any other specific binding means, for example, sequence-specific orientation in the major groove of the DNA double-helix, or any other specific binding mode. For such therapy, the oligomers of the invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington""s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition.
For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the oligomers of the invention are formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank""s solution or Ringer""s solution. In addition, the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays, for example, or using suppositories. For oral administration, the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams, as is generally known in the art.
In addition to use in therapy, the oligomers of the invention may be used as diagnostic reagents to detect the presence or absence of the target DNA or RNA sequences to which they specifically bind. Such diagnostic tests are conducted by hybridization through base complementarity or triple helix formation which is then detected by conventional means. For example, the oligomers may be labeled using radioactive, fluorescent, or chromogenic labels and the presence of label bound to solid support detected. Alternatively, the presence of a double or triple helix may be detected by antibodies which specifically recognize these forms. Means for conducting assays using such oligomers as probes are generally known.
In addition to the foregoing uses, the ability of the oligomers to inhibit gene expression can be verified in in vitro systems by measuring the levels of expression in recombinant systems.
It may be commented that the mechanism by which the specifically-binding oligomers of the invention interfere with or inhibit the activity of a target RNA or DNA is not always established, and is not a part of the invention. If the oligomer seeks, for example, a target mRNA, translation may be inhibited. In addition, by binding the target, the degradation of the mRNA message may be enhanced, or the further processing of the RNA may be inhibited. By formation of a triple helix, the transcription or replication of the subject DNA may be inhibited; furthermore, reverse transcription of infectious RNA or replication of infectious DNA is interfered with. It is also thought that the immune function may be modulated through physiological mechanisms similar to those induced by double-stranded RNA as exemplified by the xe2x80x9campligenxe2x80x9d system or similar to those used to suppress systemic lupus erythematosus. The oligomers of the invention are characterized by their ability to target specific oligonucleotide sequences regardless of the mechanisms of targeting or the mechanism of the effect thereof.
Finally, it is understood that the oligonucleotide can be derivatized to a variety of moieties which include, intercalators, chelators, lipophilic groups, label, or any other substituent which modifies but does not materially destroy the oligomeric character of the backbone.
The following examples are intended to illustrate but not to limit the invention. In all synthesis reactions, a dry argon atmosphere was used.